Figures and data
Laser stimulation induces nociceptive escape responses in the male Drosophila.
(A) In an acrylic arena covered with glass, a freely moving male fly is tracked and targeted by a 473 nm laser beam (10 mW) on the ventral thorax. (B) Representative frames showing a jumping escape response during laser irradiation. (C) Compared with wild-type w1118 controls, painless1 and trpA11 mutants exhibit prolonged jumping latency during laser irradiation. (D) trpA11 mutants show reduced locomotor velocity compared with w1118 and painless1. (E-F) A behavioral screen was performed using UAS-Kir2.1 to silence candidate Gal4 drivers targeting distinct neurotransmitter systems. Silencing trpA1-Gal4 neurons served as a positive control and increased jumping latency. The painless-Gal4 driver was excluded because the Gal4 insertion disrupts endogenous painless function. Silencing DANs labeled by TH-C1-Gal4 or TH-D1-Gal4 increased jumping latency (E); TH-D1-Gal4 also reduced walking velocity (F). Data were analyzed by Kruskal-Wallis test. Significance: ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Anatomical tracing implicate dopaminergic neurons in nociception-induced escape.
(A-B) trans-Tango labeling reveals direct downstream partners of nociceptor-associated neurons. Downstream targets of (A) painless-Gal4 and (B) trpA1-Gal4 neurons were labeled by trans-Tango (magenta), and DANs were visualized by TH immunostaining (green). Colocalization indicates that multiple dopaminergic clusters (PAL, PAM, PPL1, PPM1, PPL2ab, PPL2c, PPM2, and PPM3) are labeled as direct postsynaptic targets.
Specific subsets of PPL1 and PAM dopaminergic neurons are required for nociception-induced escape.
Targeted silencing of PPL1 subsets using split-GAL4 drivers MB058B, MB060B, MB065B, MB296B, MB304B, and MB308B increased jumping latency. Silencing PAM subsets using MB213B and MB301B similarly increased jumping latency. Quantification of (A) jumping latency and (B) velocity is shown. Statistics: Kruskal-Wallis test. Significance: ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Mushroom body output neurons (MBONs) contribute to nociception-induced escape.
Silencing MBONs increased jumping latency, with the largest effects observed for MB082C, MB083C, MB298B, MB433B, and MB434B. Statistics: jumping latency was analyzed by Kruskal-Wallis test; velocity was analyzed by one-way ANOVA where indicated. Significance: ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Working model of a MB–centered circuit for nociception-induced escape jumping.
Noxious stimuli detected through painless- and trpA1-dependent sensory pathways are proposed to recruit defined subsets of DANs labeled by TH-C1-GAL4, TH-D1-GAL4, and MB compartment-specific split-GAL4 drivers. These DANs (PPL1, blue; PAM, orange) innervate discrete MB compartments and are hypothesized to modulate compartmental MB computations to shape the output of MB output neurons (MBONs; black) that control escape-jump latency. This schematic integrates (i) behavioral silencing screens identify compartments required for normal escape performance and (ii) trans-Tango anterograde transsynaptic mapping was used to nominate downstream MBON connectivity. Arrow thickness represents Behavioral Potency Level (behavioral impact), defined as the mean escape-jump latency obtained across all driver lines targeting a given compartment, binned into high, moderate, or low effect tiers. Among DAN inputs, PPL1 innervation of α′1, α′3, and γ2 shows the largest behavioral impact, with additional contributions from α2, α3, and α′2 compartments. Within the PAM cluster, inputs to β2 and β′2 are most strongly associated with escape performance. For MBON outputs, inter-compartment MBONs projecting from β1 to the α lobe and from γ4 to γ1/γ2 exert the strongest effects, while outputs from β′1 and γ3 also contribute. Together, the convergence of dopaminergic drive and MBON routing nominates β1 and γ2 as candidate integration hubs: β1 couples strong PAM input with output to the α lobe, potentially reconfiguring downstream action selection, whereas γ2 acts as a convergence zone receiving PPL1 modulation and recurrent MBON-driven signals and is indispensable for the escape response.
